Hybrid Incompatibilities (His), including hybrid lethality and sterility, have been observed in interspecies crosses within numerous taxonomic groups across the phyla. The molecular mechanisms underlying these phenotypes are currently not known but a recently identified set of rapidly evolving genes in Drosophila offers an exciting opportunity to gain insight into this process. The goal of this proposal is to examine in detail one such gene, Hybrid male rescue (Hmr), which causes lethality in Drosophila melanogaster/D. simulans hybrids. The Hmr gene encodes a protein that contains conserved DNA-binding domains but is otherwise highly diverged among Drosophila species. These characteristics suggest that HMR normally functions as a species-specific transcriptional regulator and binds target genes abnormally in hybrid animals to cause their death. Previous genetic experiments suggested that Hmr may also interact with another hybrid lethal gene, maternal hybrid rescue (mhr). I will test these hypotheses in three specific aims. First, I will test the putative role of Hmr as a transcriptional regulator by examining the DNA binding pattern of HMR protein in D. melanogaster. Polytene chromosome analysis and chromatin immunoprecipitation (ChIP) will be used to identify candidate HMR target genes;these will be tested by assaying for transcriptional effects in the Hmr-null background and for genetic interactions with Hmr. Second, I will use similar methods to determine whether HMR binding is altered in hybrids. Specifically, I will test a subset of candidate target genes for normal HMR binding and assay for ectopic binding of HMR to inappropriate target sites throughout the genome. Individual targets will be tested for altered transcription levels. Third, I will investigate whether Hmr interacts with mhr. To do this, I will assay for effects of Hmr transgenes on mhr-dependent hybrid lethality and, conversely, I will test if mhr mutations affect Hmr-dependent hybrid lethality. This proposed work will be among the first molecular studies of an HI gene and will help to understand the relationship between the intraspecific function of Hmr and its role in hybrid lethality. Genomic studies have revealed the presence of rapidly evolving genes in a number of higher eukaryotes, including humans. Although the biological roles of these genes are largely unknown, they are likely to influence diverse phenotypic traits such as species-specific adaptations and predisposition or resistance to disease. This study of HI genes in D. melanogaster will provide a good genetic model for understanding the function of rapidly evolving genes and the roles they play in the development and health of humans.